Francisella tularensis is a highly infectious bacterium responsible for tularemia, a disease whose pneumonic form has potentially lethal consequences in humans. Francisella virulence depends on its ability to survive and replicate inside macrophages of the infected host. The current model of Francisella intracellular fate is initial enclosure within a phagosome, followed by escape from this phagosome and then replication in the cytoplasm, but the bacterial determinants controlling these individual stages are unknown. A Francisella pathogenicity island (FPI) has been identified as required for intracellular growth but how it contributes to Francisella virulence is not understood. We have been using cell biology-, bacterial genetics- and genomics-based approaches to further characterize Francisella intracellular trafficking, identify genes expressed at various stages of the intracellular cycle and assess their role in Francisella virulence. [unreadable] [unreadable] In a model of murine primary macrophage infection with F. tularensis Type B and Type A strains, we have previously shown that phagosomal escape occurs rapidly after phagocytosis ( 20 min) using a fluorescence microscopy-based assay to measure phagosomal integrity. We have also identified and characterized unsuspected, post replication events during which bacteria are enclosed within a membrane-bound compartment, the Francisella-containing vacuoles (FCV), through a process involving autophagy (Checroun et al., 2006, PNAS, 103:14578). These results have extended our knowledge of the intracellular trafficking of Francisella and indicate that this bacterium can control macrophage autophagic processes, which is an important component of the innate immune response to cytosolic pathogens. To extend our findings and further investigate how virulent Francisella respond to innate immune responses, we have developed a human blood-derived macrophage infection model, to complement our murine model of macrophage-Francisella interactions. We have investigated if and how macrophage activation with ?-IFN, which plays a critical role in controlling Francisella infection, modifies the intracellular cycle of the pathogen. In both murine and human macrophages, ?-IFN preactivation did not prevent Francisella escape from their initial phagosome, nor initiation of replication. However, ?-IFN preactivation decreased the extent of bacterial replication, suggesting a ?-IFN-dependent cytosolic control of bacterial replication, a mechanism that is currently under investigation.[unreadable] [unreadable] In our efforts to identify Francisella genes that are important for intracellular pathogenesis, we have worked in collaboration with the RTB/RTS Genomics Unit at RML to establish the intracellular transcriptome of the Type A virulent F. tularensis subsp. tularensis Schu-S4 strain. The rationale for such an approach is that genes that are essential to intracellular survival and replication of Francisella should be upregulated inside macrophages, hence identifiable through transcriptional profiling of intracellular bacteria. To validate this approach, preliminary work using quantitative PCR has been performed to characterize the expression patterns of genes from the Francisella Pathogenicity Island (FPI), which are known to be involved in intracellular growth. Such genes are induced intracellularly and their expression levels vary during the intracellular cycle and between strains of differential virulence. We have optimized our procedures to isolate and amplify quality bacterial RNA from infected murine macrophages in sufficient amounts to obtain kinetic, transcriptional profiles of intracellular bacteria using the custom-made Affymetrix RML GeneChip II. The analysis of the microarray data, which is ongoing and requires additional validation, has allowed us to characterize the genetic response of Francisella at the various stage ot its intracellular cycle and identify candidate genes involved in intracellular pathogenesis, based on their induction and their particular expression profiles inside macrophages.